The TGF-b family of ligands signal through a unique heteromeric receptor complex distinguished by its serine-threonine kinase activity. One signal transduction pathway from these receptors involves a novel family of proteins termed Smads, which are related to Drosophila Mad, a cytoplasmic mediator of signals from a TGF-b family ligand, dpp. We have demonstrated that one of these, Smad4, identical to a candidate tumor suppressor for pancreatic cancer, DPC4, can restore TGF-b signal transduction pathways in tumor cells null for Smad4. By constructing chimeric and mutated forms of Smad4, we have shown that the ligand- dependency resides in its unique proline-rich linker and N-terminal domain inserts. Ongoing studies are aimed at characterization of the functional activity associated with these domains. Other studies are focused on characterization of two novel clones we have isolated from a yeast two-hybrid system using Smad1 as bait. The proteins show no identifiable enzymatic motifs or homology to existing proteins. They suggest additional complexity to these signal transduction pathways in that they inhibit Smad-induced reporter gene activity. We are currently characterizing the interaction patterns and mechanisms of action of these proteins. In other studies, we have demonstrated that Smads may mediate cross-talk between serine-threonine kinase receptors and certain tyrosine kinase receptors, in that both HGF and EGF can phosphorylate Smad1 and Smad2 in cells lacking TGF-b receptors. HGF can also stimulate nuclear translocation of Smad2, suggesting that the overlapping activities of TGF-b and HGF in activation of certain target genes such as tissue inhibitor of metalloproteinases (TIMP) and plasminogen activator inhibitor-1 (PAI-1) are mediated by a Smad-dependent pathway. To delineate more fully the extent of Smad-mediated pathways in transduction of signals from TGF-b family ligands, we have generated mice null for Smad1 and plan to create transgenic mice expressing dominant Smad1 and 2 under control of tissue-specific promoters. The Smad1 null mice die between days 8 and 9 of embryogenesis, demonstrating a critical signaling role for this molecule in early development. We plan to backcross transgenic mice expressing dominant negative Smads with mice heterozygous for Smad1 and for Smads 2, 3, and 4 (obtained collaboratively from Chuxia Deng) to enhance the phenotype of compromised Smad activity.